Expanding homogeneous culture of human primordial germ cell-like cells maintaining germline features without serum or feeder layers

Stem Cell Reports. 2022 Mar 8;17(3):507-521. doi: 10.1016/j.stemcr.2022.01.012. Epub 2022 Feb 10.

Abstract

In vitro expansion of human primordial germ cell-like cells (hPGCLCs), a pluripotent stem cell-derived PGC model, has proved challenging due to rapid loss of primordial germ cell (PGC)-like identity and limited cell survival/proliferation. Here, we describe long-term culture hPGCLCs (LTC-hPGCLCs), which actively proliferate in a serum-free, feeder-free condition without apparent limit as highly homogeneous diploid cell populations maintaining transcriptomic and epigenomic characteristics of hPGCLCs. Histone proteomics confirmed reduced H3K9me2 and increased H3K27me3 marks in LTC-hPGCLCs compared with induced pluripotent stem cells (iPSCs). LTC-hPGCLCs established from multiple human iPSC clones of both sexes were telomerase positive, senescence-free cells readily passaged with minimal cell death or deviation from the PGC-like identity. LTC-hPGCLCs are capable of differentiating to DAZL-positive M-spermatogonia-like cells in the xenogeneic reconstituted testis (xrTestis) organ culture milieu as well as efficiently producing fully pluripotent embryonic germ cell-like cells in the presence of stem cell factor and fibroblast growth factor 2. Thus, LTC-hPGCLCs provide convenient access to unlimited amounts of high-quality and homogeneous hPGCLCs.

Keywords: embryonic germ cells; feeder-free culture; germline reprogramming; human endogenous retroviruses; induced pluripotent stem cells; primordial germ cell-like cells; primordial germ cells; serum-free culture; xrTestis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Differentiation
  • Cells, Cultured
  • Feeder Cells
  • Female
  • Germ Cells*
  • Humans
  • Induced Pluripotent Stem Cells*
  • Male